ObjectiveTo investigate the expressions and clinical significance of human telomerase reverse transcriptase (hTERT) mRNA and γglutamyl transpeptidase mRNA-H (GGT mRNA-H) in the peripheral blood of small hepatocellular carcinoma (HCC) patients. MethodsThe expressions of hTERT mRNA and GGT mRNA-H were detected in the peripheral blood of thirty patients with small HCC by RT-PCR, eighteen patients with benign liver diseases, and twelve normal volunteers. ResultsThe positive rate of hTERT mRNA and GGT mRNA-H expression in patients with small HCC were 80.0% (24/30) and 46.7%(14/30), respectively. In patients with hepatitic cirrhosis the positive rate of hTERT mRNA expression was 33.3% (6/18), while the expression of GGT mRNA was not detected. Both the expressions of hTERT mRNA and GGT mRNA-H were negative in all normal volunteers. The combination analysis of hTERT mRNA and GGT mRNA-H expression achieved positive rate of 86.7% in the diagnosis of small HCC, which was significantly higher than the positive rate of AFP (26.7%)， Plt;0.05. ConclusionThe hTERT mRNA and GGT mRNA-H are significantly expressed in small HCC patients, the combination analysis of hTERT mRNA and GGT mRNA-H seems to be useful in the early diagnosis of small HCC.
Objective To investigate the role of β-catenin gene in breast tumorigenesis by detecting mutation and expression of β-catenin gene in breast hyperplasia and breast cancer. Methods Mutation and expression of β-catenin gene in 42 breast cancer, 15 simple hyperplasia and 15 atypical hyperplasia were detected by polymerase chain reaction-single strand conformation polymorphism and immunohistochemistry. Results Normal expression of β-catenin occurred in tissue of breast simple hyperplasia. The rate of abnormal expression of β-catenin in tissue of breast atypical hyperplasia and breast cancer were 26.7% （4/15） and 59.5% （25/42）, respectively, which were higher than that of simple hyherplasia tissue (P＜0.05). And there was a markedly difference between the atypical hyperplasia tissue and breast cancer tissue (P＜0.05). Mutation of β-catenin gene wasn’t detected in this three kinds of tissues. Conclusion Abnormal expression of β-catenin plays an important role in human breast tumorigenesis, reason of abnormal expression of β-catenin isn’t mutation of β-catenin gene. Expression of β-catenin can be regulated by other mechanisms.
【Abstract】Objective To explore the changes of expression of AFP mRNA in human hepatocellular carcinoma （HCC） tissues after oral Xeloda therapy.Methods Total RNA was extracted from HCC tissue samples collect after operation and nested reverse transcription polymerase chain reaction （RT-nested PCR） assay was performed to determine the expression of AFP mRNA in this study.Results The final product of AFP mRNA amplified by RT-PCR was 174 bp and by RT-nested PCR was 101 bp. The AFP mRNA is positive in 12 of 21 patients (positive rate 57.14%) amplified by RT-nested PCR assay in Xeloda treatment group which is much lower than control group: 18 of 20 patients (positive rate 90.00%),P＜0.05.The serum AFP value of Xeloda treatment group 〔(23.2±12.8) μg/L〕 is much lower than that of control group 〔(39.6±24.3) μg/L〕 four weeks after operation (P＜0.05). However, There was no difference between two groups in serum AFP value before operation.Conclusion Xeloda can effectively suppress the expression of AFP mRNA in human HCC tissues and lower it’s product serum AFP value.The clinical application of Xeloda in HCC patients deserve further study.
Objective To study the expression and significance of multidrug resistance-associated protein (MRP) gene in hepatocellular carcinoma (HCC). Methods Reverse transcription polymerase chain reaction (RT-PCR) assay was used to detect the expression of MRP mRNA in 25 fresh specimens of the primary HCC and its surrounding liver tissues. Immunohistochemistry LSAB technique was adopted to test MRP in 60 HCC specimens. The drug sensitivity was also tested by flow cytometry.Results The positive expression rates of MRP mRNA and MRP protein in primary HCC were 44.00%(11/25) and 45.00%(27/60) respectively. All the intensity of expression was low, but significant higer than its surrouding liver tissues (P＜0.05). The intensity and expression rate of MRP protein in 5 recurrent HCC had a tendency to increase. There was a correlation between the expression of MRP mRNA and MRP protein in 25 patients using RT-PCR and immunohistochemistry assay (Plt;0.05). Detected by flow cytometry, the average sensitivity of drugs in vitro of 60 HCC sp-cimens were 5-FU （15.80±7.63）%，DDP（18.45±9.59）%，ADM（17.95±7.99）%，MMC（16.60±8.69）% and CTX（17.40±10.14）%. Only 5FU and ADM were significantly affected by the expression of MRP protein (Plt;0.05).Conclusion The expression of MRP in primary HCC may be one of the important mechanisms of the intrinsic and acquired drug resistance in HCC. To study the expression of MRP could give a predictive value in HCC chemotherapy.
Objective To evaluate the potential of specific mRNA marker keratin 19(K19) to detect micrometastasis by reverse transcriptase polymerase chain reaction (RT-PCR) .Methods One hundred and ninty four regional lymph nodes harvested from 6 cases of benign diseases, 4 cases of breast carcinoma, 5 cases of gastric carcinoma and 12 cases of colorectal carcinoma patients were examined by conventional pathology and amplifying tissue specific K19 mRNA by RT-PCR separately, then the two methods were compared with each other. Results None of the 34 lymph nodes which were pathological metastasis-negative from benign diseases expressed K19 mRNA by RT-PCR, all of the 28 regional lymph nodes which were pathological metastasis-positive from malignant cases showed trains of K19 mRNA by RT-PCR. Of the 132 lymph nodes which were pathological metastasis-negative from malignant cases, 11 lymph nodes were detected with micrometastasis by genetic diagnosis.Conclusion Genetic diagnosis of lymph node micrometastasis is more sensitive than conventional pathology and has diagnostic value and merits further study.
To investigate the mRNA expression of nm23-H1 gene in human liver tumor. In tumor and corresponding nontumoral liver specimens from 20 patients, nm23-H1 mRNA were examined by reverse transcriptionpolymerase chain reaction (RT-PCR) method with specific primers. Results: The primers designed in this study could amplified nearly entire coding sequence of nm23-H1 gene. All the samples showed positive expression of nm23-H1 mRNA, indicating there was no expression loss or obvious alteration. Conclusions: The achievement of RT-PCR method lays foundation for quantitative gaugement of nm23-H1 mRNA in liver tumor.
The aim of the this study was to search for bacterial DNA sequences in cholesterol gallstones with negative bacterial culture by NP-PCR technique. Bacterial gene fragments were amplified in vitro from DNA which were extracted from cholesterol gallstones in gallbladder for identifying the existence of bacteria. The gallbladder gallstones of 30 patients were analysed. Bacterial DNA was found in the stones of 26 patients, indicating that most cholesterol gallstones harbor bacterial DNA.
We have devised a highly sensitive, specific, and quantitative assay for multidrug resistance (mdr1) mRNA expression based on the reverse transcription-polymerase chain reaction (RT-PCR). mdr1 mRNA levels were detected in 30 human primary hepatocellular carcinoma (PHC) tissue and adjacent liver tissue. Five of the patients had received chemotherapy before hepatectomy. The results show that the level of expression of mdr1 gene is higher in tumor tissue than in adjacent liver tissue. mdr1 gene is overexpressed in PHC after chemotherapy. Furthermore, mdr1 gene expression in the treated tumor adjacent liver tissue is higher than that in untreated tumor adjacent liver tissue. Our results indicated that overexpression of mdr1 gene may be responsible for the intrinsic and acquired drug resistance of PHC.
Objective To detect the expression of forkhead box P3 （FOXP3 ）gene in esophageal squamous cell carcinoma(ESCC) and provide a new basis for immunotherapy of esophageal cancer. Methods Based on fluorescent TaqMan methodology, a realtime quantitative reverse transcription polymerase chain reaction (RT-PCR) for detecting the expression of FOXP3 was set up. In this method, a cloning vector pMD 18-T-FOXP3 was constructed as a standard plasmid. The specific expression of FOXP3 in 42 patients with ESCC and 30 healthy controls were measured by using GeneAmp 7500 Sequence Detection Systems. Results FOXP3 mRNA copy number in ESCC was significantly higher than that in healthy control tissue ［(72.20±23.10)×104copy/μg RNA vs.(0.68±0.34)×104 copy/μg RNA；Plt;0.05］. Conclusion A realtime quantitative RT-PCR method for detecting the expression of FOXP3 gene in ESCC has been successfully established. The expression level of FOXP3 is increased in ESCC compare with healthy controls.